Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma.

BACKGROUND: The clinical outcome is generally positive for patients with node-negative breast carcinoma (i.e., those who do not have detectable metastases in the lymph nodes) who have been treated with surgery or surgery plus radiation therapy. In about 30% of the patients, however, the disease recurs, and they are at risk of death. Determination of valid new prognostic indicators would improve the ability to identify patients at high risk of recurrence. Breast cancer can entail substantial development of new blood vessels within the tumor tissue, and it is known that the growth and metastasis of solid tumors are dependent on such angiogenesis. The conversion of tumor cells to an angiogenic phenotype may be preceded by a change in the balance of angiogenic growth factors and angiogenesis inhibitors. PURPOSE: This study was conducted to determine if the levels of vascular endothelial growth factor (VEGF) protein, a potent endothelial growth factor and mediator of vascular permeability and angiogenesis, measured in the primary tumors of women with node-negative breast cancer are associated with known prognostic factors and patient survival. METHODS: By use of a selective enzymatic immunoassay, levels of VEGF protein were measured in cytosolic extracts of primary tumor tissue surgically obtained from 260 women with node-negative breast carcinoma who had been treated with surgery with or without radiation therapy but not with adjuvant therapy and who had been followed for a median time of 66 months. The relationships between VEGF concentrations and other prognostic dichotomous variables or clinical outcome were tested by the use of the Kolmogorov-Smirnov test and univariate and multivariate Cox analyses, respectively. The relationship between VEGF and hormone receptors (i.e., those for estrogen and progesterone) was examined by the use of Spearman's correlation analyses. All P values resulted from the use of two-sided statistical tests. RESULTS: Tumors from 247 (95%) of the 260 patients had detectable VEGF, ranging in concentration from 5.0 to 6523 pg/mg protein (median, 126.25 pg/mg protein). No statistically significant associations were found between VEGF and the other prognostic factors (e.g., age, menopausal status, histologic tumor type, tumor size, and hormone receptors) examined. Levels of VEGF were found to be prognostic for both relapse-free and overall survival in univariate and multivariate analyses (likelihood ratio tests; all four P values < .001). In the multivariate analysis, the first-order interaction term of VEGF and estrogen receptor was also prognostic for overall survival (likelihood ratio test; P = .05). CONCLUSIONS: The results show that cytosolic levels of VEGF in tumor tissue samples are indicative of prognosis for patients with node-negative breast carcinoma.

Lipid peroxidation in the liver of carcinogen-resistant rats.

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, alpha-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.

Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides.

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.

Progesterone induces vascular endothelial growth factor on retinal pigment epithelial cells in culture.

Diabetic retinopathy is known to frequently deteriorate during pregnancy but the cause remains obscure. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a potent vascular endothelial cell mitogen which is mainly up-regulated by hypoxia, and is closely associated with the development and progression of diabetic retinopathy. To examine the influence of the drastic hormonal alterations during pregnancy on the worsening of diabetic retinopathy, we examined the effects of estradiol (E2) and progesterone (P4) on the production of VEGF/VPF in bovine retinal pigment epithelial cells in culture. The VEGF/VPF production was significantly elevated (214.5 +/- 28.3 ng/g protein, P < 0.01) by 48 h of exposure to a high concentration of P4(10 microM), which is still within the physiological range during pregnancy, compared to that of the control group (147.7 +/- 17.9 ng/g protein). However, E2 significantly stimulated the production of VEGF/VPF only at concentrations (100 microM) much higher than normally encountered during pregnancy. These two hormones were not observed to have a synergistic effect, at least at physiological concentrations. As the increase in serum P4 levels during pregnancy is reported to be greater in pregnant diabetic patients with progressive retinopathy, our findings suggest that P4 may contribute to the worsening of diabetic retinopathy during pregnancy by up-regulating intraocular VEGF levels.

An anti-human VEGF monoclonal antibody, MV833, that exhibits potent anti-tumor activity in vivo.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor for tumor angiogenesis and growth. We previously established the immunoneutralizing monoclonal antibodies (MAbs) to human VEGF, and showed that MV101 (IgG1) and MV303 (IgG2a) inhibited the growth of human solid tumor xenografts in nude mice. Then, we tried to develop another immunoneutralizing anti-VEGF MAb that exhibited more potent antitumor activity than MV101 or MV303. We obtained more than 140 clones of hybridomas that were producing anti-VEGF MAb from the mice immunized with recombinant human VEGF121. Among them, 26 clones showed the immunoneutralizing activity and MV833 possessed the most potent antitumor activity in vivo. A total of 9 i.p. administrations of 25 microg of MV833 inhibited the growth of human fibrosarcoma HT-1080 solid tumor xenografted in nude mice more potently than MV101 or MV303. Moreover, only 1 i.v. administration of 100 microg of MV833 on Day 1 after tumor inoculation also significantly inhibited the growth of HT-1080 in vivo, whereas MV101 and MV303 did not. All three MAbs inhibited the growth of human umbilical vein endothelial cells (HUVEC) induced by VEGF121 and the binding of 125I-labeled VEGF121 to HUVEC to a similar extent. The binding of MV101 and MV303 to VEGF121 was cross-competitive; however, MV833 weakly competed with the binding of MV101 to VEGF121. These findings indicated that MV833 recognized the region(s) of VEGF differently than MV101 or MV303, and this difference contributed to the superiority of antitumor activity of MV833.

Rapid measurement of urinary IL-6 by ELISA: urinary IL-6 as a marker of mesangial proliferation.

To determine whether the urinary level of interleukin 6 (IL-6) measured by enzyme-linked immunosorbent assay (ELISA) can be used as a marker of mesangial proliferation, we studied urinary levels of IL-6 in 124 patients with primary and secondary glomerulonephritis, using ELISA. Although urinary levels of IL-6 were correlated with the degree of mesangial proliferation, there was no correlation between urinary levels of IL-6 and urinary protein excretion or renal function. Urinary levels of low-molecular-weight proteins, which are parameters of tubular dysfunction, were not correlated with the urinary excretion of IL-6. These results suggest that the urinary level of IL-6 may be a useful marker for mesangial proliferation.

[The clinico-pathological significance of hematuria in diabetics].

A clinico-pathological study was performed on 154 patients with diabetes mellitus to clarify the significance of glomerular hematuria. Glomerular hematuria was observed in 26 patients (16.9%), of whom 10 had complications of IgA glomerulonephritis and one had membranous nephropathy. The remaining patients (143 cases) were divided into two groups; a hematuria group (15 cases) and a non-hematuria group (128 cases). Patients in the hematuria group showed diabetic retinopathy, hypertension, massive proteinuria and the requirement for insulin therapy more often than those in the non-hematuria group (p < 0.01, p < 0.001, p < 0.001 and p < 0.01, respectively). In addition, the serum creatinine level in the hematuria group was significantly elevated compared to that in the non-hematuria group (p < 0.01). Histologically, patients in the hematuria group exhibited advanced diffuse lesions, nodular lesions, exudative lesions, microaneurysms, crescent formation, capsular adhesion and interstitial lesions more often than those in the non-hematuria group (all, p < 0.001). Furthermore, the vascular index in the hematuria group was significantly higher than that in the non-hematuria group (p < 0.001). It is suggested that glomerular hematuria in diabetic patients indicates the presence of diabetic nephropathy at an advanced stage or coexistence of primary glomerulonephritis.

[Three cases presenting with systemic lupus erythematosus and minimal change nephrotic syndrome].

Three cases presenting with systemic lupus erythematosus (SLE) and minimal change nephrotic syndrome (MCNS) are reported in this paper. All cases were female; they abruptly developed nephrotic syndrome at the age of 30, 11 and 23 years, respectively. In Case 1, the diagnosis of SLE was based on fever, butterfly rash, Raynaud's phenomenon, leukopenia, lymphopenia, hypocomplementemia, a high titer of anti-DNA antibodies, positive DNA and LE test, and the presence of anti-nuclear antibodies (speckled pattern). In Case 2, the diagnosis was based on butterfly rash, central nervous system involvement, lymphopenia, hypocomplementemia, a positive LE cell phenomenon, a high titer of anti-DNA antibodies and a positive DNA test. In Case 3, the diagnosis was based on photosensitivity, alopecia, lymphopenia, hypocomplementemia, a high titer of anti-DNA antibodies, a positive DNA test and a positive LE cell phenomenon. In these three cases, initial symptoms were puffy face and pretibial edema which occurred suddenly. These symptoms disappeared completely after either corticosteroid therapy or a combination therapy using corticosteroids and immunosuppressive drugs. These patients took a favorable course and no aggravation was noted in the findings of urinalysis and renal functions. In two of these cases, the diagnostic criteria for SLE were satisfied, but the remaining patient fulfilled only three criteria except for renal disorder. In each of these cases, minor glomerular abnormalities were disclosed by renal histology. It seems likely that SLE was complicated by MCNS in these cases. From these cases, it is suggested that there is a possibility of immunological abnormalities associated with SLE and MCNS.

[An autopsy case of systemic lupus erythematosus complicating leukocytosis, amegakaryocytic thrombocytopenia, interstitial pneumonitis, and pleulitis].

A 52-year-old female was admitted to our hospital in August 1988, for evaluation of purpura and gingival bleeding. Hematologic examination showed mild leukocytosis (12,400/microliter) and severe thrombocytopenia (1,000/microliter). On bone marrow examination, megakaryocyte count was normal and the number of myeloblasts was increased (7.2%). Serological examination was positive for anti-nuclear antibody and anti-DNA antibody. She was diagnosed as having idiopathic or autoimmune thrombocytopenia, and received thrombocyte transfusion and gamma-globulin administration. Hematologic values improved temporarily, but leukocytosis and thrombocytopenia recurred. On the 22nd hospital day, leukocytes increased to 49,300/microliter and thrombocytes decreased to 10,000/microliter. Bone marrow myeloblasts were also increased to 18.8%, and she was suspected of having myelodysplastic syndrome. Then, hematologic values improved simultaneously, and she was discharged in November 1988. After the discharge, leukocyte count ranged from 6,000 to 16,500/microliter, but the number of bone marrow myeloblasts was normal. However, transient thrombocytopenia appeared in association with decrease or absence of bone marrow megakaryocytes and rise of platelet associated-IgG, (PA-IgG) to 99.6 ng/10(7) cells. From September to December 1989, she complained of fever, morning stiffness, multiple arthralgia, and oral ulcer. On serological findings, she was positive for LE cell. Therefore, she was diagnosed as having systemic lupus erythematosus (SLE). In January 1990, she had a high grade fever and dyspnea. Bilateral pleuritis and interstitial pneumonitis were shown on the chest roentgenogram. She received gamma-globulin administration, methylprednisolone pulse therapy, and mechanical ventilation. However, hypoxia developed rapidly, and she died of respiratory failure. Autopsy revealed severe interstitial pneumonitis, fibrinous pleuritis, fibrinous pericarditis, and vasculitis in the arcuate artery of the kidney. This is the first report of SLE complicating thrombocytopenia associated with decrease of megakaryocytes and rise of the PA-IgG, and severe leukocytosis associated with increased bone marrow myeloblasts.

Anti-nuclear RNP antibodies in two sisters.

Two sisters with clinical elements of mixed connective tissue disease were found to have anti-nuclear RNP (nRNP) antibodies. These antibodies were not found in the six other family members examined. The sisters had inherited an identical HLA haplotype A2-Bw61-Cw3-DR1 from their mother who had had Raynaud's phenomenon for several years. Analysis of peripheral lymphocyte subsets in the patients and their immediate relatives showed decreased OKT-4-positive cells in one of the patients and increased OKT-8-positive cells in both patients, their father and their brother, resulting in lower OKT 4/OKT 8 ratios in these members of the family. This is the third description of the familial occurrence of anti-nRNP antibodies and it adds further evidence for the implication of genetic factors in the development of anti-nRNP antibodies.

Vascular endothelial growth factor is induced by long-term high glucose concentration and up-regulated by acute glucose deprivation in cultured bovine retinal pigmented epithelial cells.

Vascular endothelial growth factor (VEGF) is closely correlated to diabetic retinopathy. Its basal production in three types of cultured retinal cells (endothelial cells, pericytes and retinal pigment epithelial cells; RPE) was examined. RPE production of VEGF was markedly higher than the rest of the cells. VEGF production in RPE was significantly elevated by 10-day, but not by 1- or 3-day exposure to 16.5 mM glucose compared to a 5.5 mM glucose group. Transient deterioration of diabetic retinopathy is frequently observed during rapid correction of glycemic control. To determine whether VEGF is up-regulated following a sharp drop in the glucose concentration or not, we examined the changes in VEGF production in RPE before and after a sudden drop in the glucose concentration. VEGF production was significantly increased by a glucose concentration decrease from 5.5 to 0.5 mM, but not by a decrease from 33 or 16.5 to 5.5 mM. These findings suggest that up-regulation of VEGF may contribute to the development of diabetic retinopathy and its worsening by hypoglycemia.

Sensitive chemiluminescence enzyme immunoassay for vascular endothelial growth factor/vascular permeability factor in human serum.

A sandwich chemiluminescence enzyme immunoassay for measuring the level of VEGF/VPF in serum was constructed. The detectability of the assay is very low (1.0 pg/ml) and the measurable range of the assay was very wide (1-1000 pg/ml). The assay showed that the average level of VEGF/VPF in human sera from healthy blood donors was approximately 19 pg/ml.